Iron is both essential and toxic. Malregulation of iron metabolism resulting in decreased iron acquisition leads to anemia, while excessive iron accumulation leads to iron overload disease. Ferroportin (Fpn) is the only known iron exporter and understanding the regulation of Fpn is essential to understanding iron homeostasis. Fpn functions as a homodimer and missense mutations in Fpn result in a dominantly inherited iron-overload disease. We will identify domains in Fpn important for dimer formation and for the proper trafficking of Fpn to the cell surface. Systemic iron physiology is regulated by the binding of the peptide hormone hepcidin to Fpn leading to Fpn internalization and degradation. We previously identified the hepcidin-binding domain on Fpn. We will determine which amino acids in hepcidin are important for hepcidin conformation and Fpn binding. We will resolve the structure of hepcidin bound to the hepcidin-binding domain on Fpn by NMR. We determined that hepcidin induces the binding of Jak2 to Fpn. We will determine the binding site on Fpn for Jak2 and how Fpn monomers interact to permit Jak2 binding and activation. We will also determine how hepcidin induces the binding of hsp70 to Fpn and the role of hsp70 in Fpn internalization. We discovered that cellular iron deprivation leads to hepcidin-independent internalization of Fpn that is dependent on ubiquitination of Fpn. We determined that Nedd4-2 is the ubiquitin ligase responsible for hepcidin-independent internalization of Fpn, as well the ubiquitin ligase responsible for hepcidin-dependent internalized Fpn entry into the multivesicular body. Our data suggests that Nedd4-2 does not bind to Fpn directly. We will identify the proteins that mediate the effects of Nedd4-2 on Fpn. We will test the hypothesis that hepcidin- independent ubiquitination is a response to altered Fpn conformation. Using mice that have a FLOXED Nedd4-2, we will determine the role of iron starvation-induced Fpn degradation in cell and organ physiology. These studies will define the regulation of Fpn in normal and disease states and provide new opportunities for the treatment of both iron-overload disorders and anemia due to increased serum hepcidin. PUBLIC HEALTH RELEVANCE: Iron is an essential element but is toxic in excess. Malregulation of iron transport results in tissue injury, either from iron deprivation or overload. We will characterize iron transport through ferroportin, the only known mammalian iron exporter. We will determine the domains in ferroportin important for dimer formation and iron transport. We will determine how the hormone hepcidin regulates ferroportin internalization and the mechanism of hepcidin-independent ferroportin internalization. Our studies will provide information on how alterations in ferroportin result in disease and create new opportunities to manage and diagnose human diseases due to altered iron metabolism.